Human gene therapy is an approach to treating human disease that is based on the modification of gene expression in cells of the patient. It has become apparent over the last decade that the single most outstanding barrier to the success of gene therapy as a strategy for treating inherited diseases, cancer, and other genetic dysfunctions is the development of useful gene transfer vehicles. For optimal use, gene transfer vehicles must have the following characteristics: (1) the ability to transduce a selected gene into a nondividing cell efficiently; (2) the ability to transduce with specificity into a wide variety of target cells; (3) the ability to provide transient or long-term, stable transgene expression; and (4) the ability to present no health risk to the recipient. It is equally important from a practical/technical standpoint that the envisioned vectors are easy to manipulate and manufacture.
Eukaryotic viruses have been employed as vehicles for somatic gene therapy. Among the viral vectors that have been cited frequently in gene therapy research are adenovirus and adeno-associated virus (AAV). Adenovirus and AAV are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types. These viral vectors, however, also have inherent limitations in use as vehicles for somatic gene therapy.
Adenovirus vectors are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state. High titers (10.sup.13 plaque forming units/ml) of recombinant virus can be easily generated in 293 cells (the adenovirus equivalent to retrovirus packaging cell lines) and cryostored for extended periods without appreciable losses. The efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders Y. Watanabe, Atherosclerosis, 36:261-268 (1986); K. Tanzawa et al, FEBS Letters, 118(1):81-84 (1980); J. L. Golasten et al, New Engl. J. Med., 309(11983):288-296 (1983); S. Ishibashi et al, J. Clin. Invest., 92:883-893 (1993); and S. Ishibashi et al, J. Clin. Invest., 93:1885-1893 (1994)!. Indeed, recombinant replication defective adenovirus encoding a cDNA for the cystic fibrosis transmembrane regulator (CFTR) has been approved for use in at least two human CF clinical trials see, e.g., J. Wilson, Nature, 365:691-692 (Oct. 21, 1993)!.
The primary limitation of this virus as a vector resides in the complexity of the adenovirus genome. A human adenovirus is comprised of a linear, approximately 36 kb double-stranded DNA genome, which is divided into 100 map units (m.u.), each of which is 360 bp in length. The DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication. The gene products are organized into early (E1 through E4) and late (L1 through L5) regions, based on expression before or after the initiation of viral DNA synthesis see, e.g., Horwitz, Virology, 2d edit., ed. B. N. Fields, Raven Press, Ltd. New York (1990)!.
A human adenovirus undergoes a highly regulated program during its normal viral life cycle Y. Yang et al, Proc. Natl. Acad. Sci. USA, 91:4407-4411 (1994)!. Virions are internalized by receptor-mediated endocytosis and transported to the nucleus where the immediate early genes, E1a and E1b, are expressed. Because these early gene products regulate expression of a variety of host genes (which prime the cell for virus production) and are central to the cascade activation of early delayed genes (e.g. E2, E3, and E4) followed by late genes (e.g. L1-5), first generation adenovirus vectors for gene therapy focused on the removal of the E1 domain. This strategy was successful in rendering the vectors replication defective, however, in vivo studies revealed transgene expression was transient and invariably associated with the development of severe inflammation at the site of vector targeting S. Ishibashi et al, J. Clin. Invest., 93:1885-1893 (1994); J. M. Wilson et al, Proc. Natl. Acad. Sci., USA, 85:4421-4424 (1988); J. M. Wilson et al, Clin. Bio., 3:21-26 (1991); M. Grossman et al, Som. Cell. and Mol. Gen., 17:601-607 (1991)!.
Adeno-associated viruses (AAV) have also been employed as vectors for somatic gene therapy. AAV is a small, single-stranded (ss) DNA virus with a simple genomic organization (4.7 kb) that makes it an ideal substrate for genetic engineering. Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep62 and rep40) are involved in replication, rescue and integration of the AAV genome. The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5' and 3' ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. Of importance for the development of AAV vectors, the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp.155-168 (1990)!. It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome.
The AAV life cycle is biphasic, composed of both latent and lytic episodes. During a latent infection, AAV virions enter a cell as an encapsidated ssDNA, and shortly thereafter are delivered to the nucleus where the AAV DNA stably integrates into a host chromosome without the apparent need for host cell division. In the absence of helper virus, the integrated ss DNA AAV genome remains latent but capable of being activated and rescued. The lytic phase of the life cycle begins when a cell harboring an AAV provirus is challenged with a secondary infection by a herpesvirus or adenovirus which encodes helper functions that are recruited by AAV to aid in its excision from host chromatin B. J. Carter, cited above!. The infecting parental ssDNA is expanded to duplex replicating form (RF) DNAs in a rep dependent manner. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or - ss DNA genomes following cell lysis.
Progress towards establishing AAV as a transducing vector for gene therapy has been slow for a variety of reasons. While the ability of AAV to integrate in quiescent cells is important in terms of long term expression of a potential transducing gene, the tendency of the integrated provirus to preferentially target only specific sites in chromosome 19 reduces its usefulness. Additionally, difficulties surround large-scale production of replication defective recombinants. In contrast to the production of recombinant retrovirus or adenovirus, the only widely recognized means for manufacturing transducing AAV virions entails co-transfection with two different, yet complementing plasmids. One of these contains the therapeutic or reporter minigene sandwiched between the two cis acting AAV ITRs. The AAV components that are needed for rescue and subsequent packaging of progeny recombinant genomes are provided in trans by a second plasmid encoding the viral open reading frames for rep and cap proteins. The cells targeted for transfection must also be infected with adenovirus thus providing the necessary helper functions. Because the yield of recombinant AAV is dependent on the number of cells that are transfected with the cis and trans-acting plasmids, it is desirable to use a transfection protocol with high efficiency. For large-scale production of high titer virus, however, previously employed high efficiency calcium phosphate and liposome systems are cumbersome and subject to inconsistencies.
There remains a need in the art for the development of vectors for gene therapy which overcome the disadvantages of the known vector systems.